The cell division cycle is one of the most fundamental processes in biology which, in multicellular organisms, ensures the controlled generation of cells with specialized functions. Under normal growth conditions, cell proliferation is tightly regulated in response to diverse intra- and extracellular signals. This is achieved by a complex network of proto-oncogenes and tumor-suppressor genes that are components of various signal transduction pathways. Activation of a proto-oncogene(s) and/or a loss of a tumor suppressor gene(s) can lead to the unregulated activity of the cell cycle machinery. This, in turn, will lead to unregulated cell proliferation and to the accumulation of genetic errors which ultimately will result in the development of cancer (Pardee, Science 246:603-608, 1989).
In the eukaryotic cell cycle a key role is played by the cyclin-dependent kinases (CDKs). Cdk complexes are formed via the association of a regulatory cyclin subunit and a catalytic kinase subunit. In mammalian cells, the combination of the kinase subunits (such as cdc2, CDK2, CDK4 or CDK6) with a variety of cyclin subunits (such as cyclin A, B1, B2, D1, D2, D3 or E) results in the assembly of functionally distinct kinase complexes. The coordinated activation of these complexes drives the cells through the cell cycle and ensures the fidelity of the process (Draetta, Trends Biochem. Sci. 15:378-382, 1990; Sherr, Cell 73:1059-1065, 1993). Each step in the cell cycle is regulated by a distinct and specific cyclin-dependent kinase. For example, complexes of Cdk4 and D-type cyclins govern the early G1 phase of the cell cycle, while the activity of the CDK2/cyclin E complex is rate limiting for the G1 to S-phase transition. The CDK2/cyclin A kinase is required for the progression through S-phase and the cdc2/cyclin B complex controls the entry into M-phase (Sherr, Cell 73:1059-1065, 1993).
The CDK complex activity is regulated by mechanisms such as stimulatory or inhibitory phosphorylations as well as the synthesis and degradation of the kinase and cyclin subunit themselves. Recently, a link has been established between the regulation of the activity of cyclin-dependent kinases and cancer by the discovery of a group of CDK inhibitors including the p27Kip1, p21Waf1/Cip1 and p16Ink4/MTS1 proteins. The activity of p21Waf1/Cip1 is regulated transcriptionally by DNA damage through the induction of p53, senescence and quiescence (Harper et al., Cell 75:805-816, 1993). The inhibitory activity of p27Kip1 is induced by the negative growth factor TGF-xcex2 and by contact inhibition (Polyak et al., Cell 78:66-69, 1994). These proteins, when bound to CDK complexes, inhibit their kinase activity, thereby inhibiting progression through the cell cycle. Although their precise mechanism of action is unknown, it is thought that binding of these inhibitors to the CDK/cyclin complex prevents its activation. Alternatively, these inhibitors may interfere with the interaction of the enzyme with its substrates or its cofactors.
While p21Waf1/Cip1 and p27Kip1 inhibit all the CDK/cyclin complexes tested, p16Ink4/MTS1, p15, p18 and p19 block exclusively the activity of the CDK4/cyclin D and CDK6/cyclin D complexes in the early G1 phase (Serrano et al., Nature 366:704-707, 1993), by either preventing the interaction of Cdk4 and Cyclin D1, or indirectly preventing catalysis. As mentioned above, the p21Waf1/Cip1 is positively regulated by the tumor suppressor p53 which is mutated in approx. 50% of all human cancers. p21Waf1/Cip1 may mediate the tumor suppressor activity of p53 at the level of cyclin-dependent kinase activity. p16Ink4/MTS1 is the product of a tumor suppressor gene localized to the 9p21 locus, which is frequently mutated in human cancer cells.
Of all the various kinases, the CDK4/cyclin D complexes are known to play an important role in regulating cell cycle progression in early G1. These complexes function as integrators of various growth factor-induced extracellular signals and as a link between the different signal transduction pathways and other cyclin-dependent kinases. The expression of the cyclin D1 positive regulatory subunit, is deregulated by gene translocations, retroviral insertions and amplifications in parathyroid adenomas, lymphomas, esophageal and breast carcinomas. The targeted overexpression of cyclin D1 in the mammary epithelium of transgenic mice induces mammary adenomas and adenocarcinomas. This confirms that cyclin D1, when overexpressed, acts as an oncogene (Wang et al., Nature 369:669-671, 1994). These data supports the idea that the lack of functional p16Ink4/MTS1 or the overexpression of cyclin D1 leads to the deregulation of CDK4/cyclin D1 kinase activity and thereby contribute to uncontrolled cell proliferation.
The prominent role of CDK/cyclin kinase complexes, in particular, CDK4/cyclin D kinase complexes, in the induction of cell proliferation and their deregulation in tumors, makes them ideal targets for developing highly specific anti-proliferative agents.
In one aspect, the present invention relates to a nucleic acid comprising a nucleotide sequence encoding a chimeric polypeptide having at least two CDK-binding motifs derived from different proteins which bind to cyclin dependent kinases (CDKs). The chimeric polypeptide binds to CDKs and inhibits cell-cycle progression.
The chimeric polypeptide can be a fusion protein, or can be generated by chemically cross-linking the CDK-binding motifs.
In preferred embodiments, at least one of the CDK-binding motifs is a CDK-binding motif of a CDK inhibitor protein, such as an INK4 protein, e.g., p15, p16, p18 and p19, or a CIP protein, e.g., p21CIP1, p27KIP1, and p57KIP2. However, it will be understood that other CDK-binding motifs may be useful. Indeed, the CDK-binding motif of the INK4 proteins is characteristized by tandemly arranged ankyrin-like sequences, which sequences exist in other proteins and, for those which are able to bind a CDK, can be used to generate the subject chimeric proteins. Likewise, the CDK-binding motif can be a p21/p27 inhibitory domain of a protein which has some homology with the CIP protein family. Exemplary chimeric proteins of the present invention are designated by SEQ ID No:2, 5 and 7, and are encoded by the CDS""s designated in SEQ ID No:1, 4 and 6.
In preferred embodiments, the CDK-binding motifs of the chimeric protein have different binding specificities, relative to one and other, for cyclin dependent kinases. For instance, the chimeric protein can be generated with a CDK-binding motif from a protein which binds to and inhibits a CDK involved in progression of the cell cycle in G0 and/or G1 phase, and another CDK-binding motif from a protein which binds to and inhibits a CDK involved in progression of the cell cycle in S, G2 and/or M phase. That is, the chimeric protein will bind to and inhibit a plurality (two or more) of cyclin dependent kinases which are active in different phases of the cell-cycle.
In most embodiments, the nucleic acid will further include a transcriptional regulatory sequence for controlling transcription of the nucleotide sequence encoding the chimeric polypeptide, e.g., the transcriptional regulatory sequence is operably linked to a chimeric gene encoding the chimeric polypeptide. For example, the present invention specifically contemplates recombinant transfection systems which include: (i) a gene construct including a nucleic acid encoding a chimeric polypeptide comprising CDK-binding motifs from two or more different proteins which bind to cyclin dependent kinases, and operably linked to a transcriptional regulatory sequence for causing expression of the chimeric polypeptide in eukaryotic cells, and (ii) a gene delivery composition for delivering the gene construct to a cell and causing the cell to be transfected with the gene construct. For example, the gene construct can be derived from a viral vector, such as an adenoviral vector, an adeno-associated viral vector or a retroviral vector. In such embodiments, the gene delivery composition comprises a recombinant viral particle. In other embodiments, the gene construct can be delivered by such means as a liposome or a poly-cationic nucleic acid binding agent. For in vivo delivery to a mammal, such as a human, the gene delivery composition will further include a pharmaceutically acceptable carrier for adminstration to an animal, and, as necessary, will be a sterile preparation and substantially free of pyrogenic agents.
The present invention also pertains to preparations of such chimeric polypeptides. e.g., polypeptides which are generated from CDK-binding motifs from two or more different proteins which bind to cyclin dependent kinases. In preferred embodiments, the chimeric polypeptide is formulated in pharmaceutically acceptable carrier for delivery to a mammal. For example, the chimeric polypeptide can be formulated in liposomal preparations.
Still another aspect of the present invention related to transgenic animals which have cells harboring a nucleic acid one of the subject fusion proteins.
Yet another aspect of the present invention relates to recombinant transfection systems, comprising
(i) a first gene construct comprising a coding sequence encoding a inhibitory polypeptide comprising at least one CDK-binding motif for binding and inhibiting activation of a cyclin dependent kinase (cdk), which coding sequence is operably linked to a transcriptional regulatory sequence for causing expression of the first polypeptide in eukaryotic cells,
(ii) a second gene construct comprising a coding sequence encoding a endotheliazation polypeptide which promotes endothelialization, and
(ii) a gene delivery composition for delivering the gene constructs to a cell and causing the cell to be transfected with the gene construct.
In preferred embodiments, the CDK-binding motif is a CDK-binding motif of a CDK inhibitor protein, such as an INK4 protein (e.g., p15, p16, p18 or p19), or a CIP/KIP protein (e.g., p21CIP1, p27KIP1, and p57KIP2). In other preferred embodiments, the CDK-binding motif comprises tandemly arranged ankyrin-like sequences, or a p21/p27 inhibitory domain.
The inhibitory polypeptide can be a fusion protein comprising CDK-binding motifs from two or more different proteins which bind to cyclin dependent kinases.
In preferred embodiments, the endothelization polypeptide stimulates endothelial cell proliferation and/or stimulates migration of endothelial cells to a wound site. For instance, the endothelization polypeptide is selected from the group consisting of angiogenic basic fibroblast growth factors (bFGF), acid fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), vascular permeability growth factor (VPF), and transforming growth factor beta (TGF-xcex2).
In preferred embodiments, the the first and second gene constructs are provided as part of a single vector, and can be provided as part of polycistronic message. Alternatively, the the fisrt and second gene constructs are provided on separate vectors.
In certain embodiments, the gene construct comprises a viral vector, e.g., an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.
In other embodiments, the gene construct is provided in a delivery composition, e.g., selected from the group consisting of a liposome and a poly-cationic nucleic acid binding agent.
The subject transfection systems can be used for treating an animal having a vascular wound characterized a breech of endothelial integrity and by excessive smooth muscle proliferation, by a method which includes administering the recombinant transfection system to the area of the vascular wound. In preferred embodiments, the subject transfection systems are used in the treatment of restenosis, and may be administered by catheter.
Still another aspect of the present invention provides a gene construct encoding a fusion protein comprising a therapeutic polypeptide sequence from an intracellular protein which alter a biological process of a cell upon intracellular localization of the fusion protein, and a transcellular polypeptide sequence for promoting transcytosis of the fusion protein across a cell surface membrane and into a cell.
In preferred embodiments, the trancellular fusion protein alters one or more such biological processes as proliferation, differentiation, cell death, gene expression, protein stability, calcium mobilzation, ion permability, phosphorylation of intracellular proteins, metabolism of inositol phosphates (IP3 and the like, diacyl glycerides, etc), and metabolism of nucleosides (such as cAMP).
The transcellular fusion protein can, by virtue of its binding to a protein or nucleic acid in the targeted cell, alter (inhibit or potentiate) protein-protein interactions or protein-nucleic acid interactions between proteins and nucleic acids endogenous to the cell.
In certain embodiments, the therapeutic polypeptide sequence can be dervied from a tumor suppressor, a transcription factor, a signal transduction protein, an antiviral protein or a metal chelating protein. For instance, the therapeutic polypeptide sequence can include a polypeptide sequence of a tumor supressor such as p53, Rb or an Rb-like protein, or a CKI protein. In other embodiments, the therapeutic polypeptide sequence includes a polypeptide sequence of a signal transduction protein, such as from tubby, a DOT protein, a Bcl protein (bcl-2, bcl-x, etc), or an IxcexaB protein.
In a preferred embodiment, the fusion protein includes at least one CDK-binding motif for binding and inhibiting activation of a cyclin dependent kinase (cdk). For instance, the CDK-binding motif is a CDK-binding motif of a CDK inhibitor protein, such as an INK4 protein (e.g., p15, p16, p18 or p19), or a CIP/KIP protein (e.g., p21CIP1, p27KIP1, and p57KIP2). In other preferred embodiments, the CDK-binding motif comprises tandemly arranged ankyrin-like sequences, or a p21/p27 inhibitory domain. In preferred embodiments, the polypeptide comprises CDK-binding motifs from two or more different proteins which bind to cyclin dependent kinases.
The transcellular polypeptide sequence can be an internalizing peptide, such as may be derived from a polypeptide selected from the group consisting of antepennepedia protein, HIV transactivating (TAT) protein, mastoparan, melittin, bombolittin, delta hemolysin, pardaxin, Pseudomonas exotoxin A, clathrin, Diphtheria toxin and C9 complement protein.
In other embodiments, the transcellular polypeptide sequence can be an accessory peptide sequence which enhances interaction of the fusion protein with a cell surface membrane, such as a peptide sequence that includes an RGD sequence.
In preferred embodiments, the gene construct comprises a viral vector, e.g., an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.
In other embodiments, the gene construct is provided in a delivery composition, e.g., selected from the group consisting of a liposome and a poly-cationic nucleic acid binding agent.
The invention also provides compositions of the fusion protein, e.g., such as may be formulated in pharmaceutical preparations.
The gene construct and the fusion protein may each be used as part of a method for treating an animal for unwanted cell proliferation, by administering the chimeric gene or the fusion protein.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).